Transposable genetic elements constitute a consid erable part of eukaryotic genome and have a marked effect on its function and structurization [1]. The pres.
ISSN 16076729, Doklady Biochemistry and Biophysics, 2009, Vol. 429, pp. 293–295. © Pleiades Publishing, Ltd., 2009. Original Russian Text © A.P. Kotnova, V.B. Salenko, N.V. Lyubomirskaya, Y.V. Ilyin, 2009, published in Doklady Akademii Nauk, 2009, Vol. 429, No. 1, pp. 120–122.
BIOCHEMISTRY, BIOPHYSICS AND MOLECULAR BIOLOGY
Structural Organization of Heterochromatin in Drosophila melanogaster: Inverted Repeats of Transposable Element Clusters A. P. Kotnova, V. B. Salenko, N. V. Lyubomirskaya, and Academician Y. V. Ilyin Received June 2, 2009
DOI: 10.1134/S1607672909060027
Transposable genetic elements constitute a consid erable part of eukaryotic genome and have a marked effect on its function and structurization [1]. The pres ence of a great number of highly diverged copies of dif ferent transposable elements in the heterochromatin of eukaryotes has long remained a mystery and was most often considered as a peculiar “cemetery” of ret rotransposons. However, recent studies showed that these heterochromatin regions harbor the clusters of transposable elements producing short RNAs, which may be then involved in the elimination of transcripts of active euchromatic copies of transposable elements [2]. There is another relevant problem that requires special attention of researchers—the involvement of associations of transposable elements in the formation of chromatin architecture. It was shown that some transposable elements (in particular, the MDG4 endogenous retrovirus of Drosophila melanogaster, which is also known as gypsy) contain regulatory sequences, such as insulators, which can influence the functioning of genes located at large distances from the transposable element [3, 4]. However, the question on the effect of transposable element clusters occur ring in the genome on the chromatin conformation and their role in the formation of epigenetic structures that can affect gene function without changing the pri mary structure of genomic DNA remains to be answered. In this study, we revealed two regions in the D. mel anogaster genome containing inverted repeats repre sented by fragments of transposable elements. Possible involvement of such structures in the regulation and functioning of the genome is discussed. In the past decade, the D. melanogaster strain G32 has been actively studied in our laboratory. The genome of this strain, along with the active and inac
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow, 119991 Russia
tive MDG4 (gypsy) variants, contains a hybrid variant [5, 6]. The genomic library of D. melanogaster strain G32 contains 24 different MDG4 copies, the majority of which are located in the heterochromatin [6]. A detailed structural analysis of these copies and their genomic environment provides a unique opportunity to study heterochromatin structure within one strain and compare it with the sequences available from databases. The sequencing of the clones from the genomic library of D. melanogaster strain G32 that contain diverged MDG4 (gypsy) copies showed that one clone, named 40A, contained a sequence with inverted repeats represented by fragments of different transpos able elements. To determine the location of the cloned sequences, we performed the search for homologous sites in the FlyBase database of the Drosophila genome. As a result, clone 40A was found to be local ized to the Xchromosome heterochromatic region (X Het: 151118–135955). In addition, successful localization of clone 4.2 of the D. melanogaster strain G32 to the heterochromatin of the left arm of chromo some 3 (3LHet: 1614362–1623973) made it possible to find one more fragment in the FlyBase database of the Drosophila genome, containing three repeats of transposable element clusters—two direct and one inverted. Figure 1 shows the schematic structure of clone 40A (15164 bp), which is represented primarily by fragments of different transposable elements. Frag ments of the same element adjoining one another are united. Of special interest is the presence of two inverted repeats, which are indicated with thin arrows below the scheme (forward repeat, 5151 bp; reverse repeat, 5168 bp). Each repeat is represented by oppo sitely directed highly rearranged transposable ele ments BS (fragments 1749–2010, 2098–3564, 3785– 4296, and 4467–5103 or 4467–5116 are united within one white arrow) and gypsy (fragments 934–1004, 1202–1660, and 4286–4398 are united within one gray arrow; fragments 4436–4505 and 4615–4912,
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KOTNOVA et al. gypsy (4934–5212) G (653–3651) G (4041–4181)
G (3867–4181)
gypsy (4436–4912)
gypsy (4436–4912) gypsy (934–4398)
gypsy (934–4398) BS (1749–5116)
BS (1749–5103)
1 kb Repeat 1
Repeat 2
Fig. 1. Clone 40A structure. Schematic representation of clone 40A from D. melanogaster strain G32. Fragments of the same diverged transposable element adjoining each other are united. Thick white arrows indicate the BS element fragments; gray arrows, the MDG4 (gypsy) fragments; and black arrows, the G element fragments. Thin arrows below the scheme indicate inverted repeats. Here and in Figs. 2 and 3, numerals in parentheses correspond to the serial numbers of nucleotides in the sequence of each transposable element.
gypsy (3824–4114) gypsy (4194–4912) gypsy (3536–3789) gypsy (4952–5400) gypsy (2772–2886) gypsy (5676–5808) gypsy (1202–1923) Doc (2503–2840) gypsy (932–1018) gypsy12 (2750–2824) X (2550–3616) gypsy12 (3276–3800) X (1711–2521) gypsy12 (3991–4453) gypsy12 (4831–5092) X (880–953) gypsy12 (5128–5492) X (4132–4290) 1 kb Repeat 1 Fig. 2. Clone 4.2 structure. Detailed structure of clone 4.2 from the D. melanogaster strain G32. Thick white arrows indicate the X element fragments; gray arrows, the MDG4 (gypsy) fragments; and black arrows, the gypsy12 fragments. The fragment of the Doc element is crosshatched with oblique lines. The thin arrow below the scheme indicates one of three repeats in the hetero chromatin in chromosome, which is present in the clone.
within the other gray arrow), as well as includes a small region of the G element (fragment 4041–4181), located between the repeats and carrying multiple deletions (shown with thick black arrows). The numeration of nucleotides is given according to the canonical sequence for each of the mentioned trans posable elements. The identity of repeats was very high: over a 5kb region they only differed in single substitutions and several deletions/insertions of single nucleotides. Figure 2 shows a detailed structure of clone 4.2, containing the fragments of elements X, MDG4 (gypsy), Doc, and gypsy12. Its length was 9612 bp. When determining the location of this fragment in the Drosophila genome, it was found that the sequence from the database that adjoins clone 4.2 contains two
more repeats of the fragment of this clone (forward and inverted), with slight variations. The schematic representation of this region, located in the hetero chromatic regions of chromosome 3 (3LHet: 1614362–1646000) is shown in Fig. 3. Fragments of the same element adjoining one another are united. As can be seen from Fig. 2, the repeated region (5676 bp long) present in clone 4.2 is represented by the following fragments of transposable elements: X element (880–953, 1711–2521, and 2550–3616) and gypsy (823–896, 932–1018, 1202–1923, 2772–2886, 3536–3789, 3824–4114, 4194–4912, 4952–5400, and 5676–5808). The numeration of nucleotides is given according to the canonical sequence for each of the mentioned transposable elements. A forward repeat 5943 bp long is located approximately 13 kb
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1360 (51–569) 1360 (3114–3409) gypsy12 (5584–6613) gypsy12 (2122–3668) X (4427–4654) Doc (2963–3038) X (1711–3616) Doc (2859–3595) gypsy (823–6558) gypsy12 (3276–7886) gypsy (823–5317) Doc (2503–2840) X (1711–3616) gypsy (923–5808) X (880–953) X (1711–3616) X (4132–4713) X (880–953) X (4132–4290)
Repeat 1
Repeat 2
Repeat 3 5 kb
Clone 4.2 Fig. 3. Structure of the Drosophila genome region 3LHet: 1614362–1646000 taken from the Flybase database. Schematic repre sentation of the Drosophila genome region 3LHet: 1614362–1646000 taken from the Flybase database. Fragments of the same diverged transposable element adjoining each other are united. Thick white arrows indicate the X element fragments; gray arrows, the MDG4 (gypsy) fragments; and black arrows, the gypsy12 fragments. The fragments of the Doc element and element 1360 are crosshatched with oblique and horizontal lines, respectively. Thin arrows below the scheme indicate repeats. The line below the scheme indicates the position of clone 4.2 from the D. melanogaster strain G32.
from it (Fig. 3). It is slightly different. First, the frag ment of the X element (880–953) is absent in it. Sec ond, there is one more small MDG4 (gypsy) repeat, which, along with the sequence 1202–1923, contains sequence 1202–1850 immediately upstream of the former (i.e., this region underwent dimerization); third, it ends with a more extended MDG4 (gypsy) fragment (5676–6558 instead of 5676–5808 in clone 4.2). There are some more insignificant dele tions and substitutions. This repeat is immediately fol lowed by the third, inverted repeat 5490 bp long, the main distinction of which from the cluster from clone 4.2 is the absence of the MDG4 (gypsy) fragment 5676–5808 and truncated variant of the previous region (the inverted repeat contains sequence 4958– 5317 instead of sequence 4952–5400 in clone 4.2). The structures discovered by us in the Drosophila genome, containing inverted repeats within transpos able element clusters are undoubtedly of interest. The presence of such sequences in the genome of the D. melanogaster strain G32 as well as in the genome from the FlyBase database is indicative of their con served nature, which is an indirect evidence for their important functions. It is known that both sense and antisense short RNAs are substrates of different groups of enzymes involved in cascades of RNA silencing reactions [2]. The presence in the genome of sequences able to simultaneously produce both short RNA variants led us to assume that these regions may function as a much more potent blocker of transposi tion of mobile elements than the extended hetero chromatic clusters devoid of such repeats. In addition, such structures may be involved in the formation of stable epigenetic relationships. The MDG4 (gypsy) DOKLADY BIOCHEMISTRY AND BIOPHYSICS
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endogenous retrovirus contains an insulator, whose fragment is present in inverted repeats of the discov ered transposable element clusters. The role of MDG4 (gypsy) insulator in the formation of epigenetic struc tures is already confirmed; however, the mechanisms of formation of insulator bodies have been insuffi ciently studied and are debated in the world scientific community. The origin of the inverted clusters of transposable elements remains to be established. ACKNOWLEDGMENTS This study was supported by the program of the President of the Russian Federation for support of young candidates of science (project no. MK 5439.2008.4), the program “Leading Scientific Schools” (project no. NSh2994.2008.4), and the Russian Foundation for Basic Research (project nos. 080400227a and 090412172ofi_M). REFERENCES 1. Churikov, N.A., Biokhimiya, 2005, vol. 70, no. 4, pp. 493–513. 2. Brennecke, J., Aravin, A.A., Stark, A., et al., Cell, 2007, vol. 128, pp. 1089–1103. 3. Gdula, D.A., Gerasimova, T.I., and Corces, V.G., Proc. Natl. Acad. Sci. USA, 1996, vol. 93, pp. 9378–9383. 4. Dorsett, D., Curr. Opin. Genet. Dev., 1999, vol. 9, pp. 505–514. 5. Lyubomirskaya, N.V., Smirnova, J.B., Razorenova, O.V., et al., Mol. Gen. Genom., 2001, vol. 265, pp. 367– 374. 6. Salenko, V.B., Kotnova, A.P., Karpova, N.N., et al., Mol Gen. Genom., 2008, vol. 279, no. 5, pp. 463–472. 2009
